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Kinetic Studies on the Effect of Skp and YaeT from Escherichia coli on the Insertion and Folding of Outer Membrane Protein A into Lipid Membranes

Kinetic Studies on the Effect of Skp and YaeT from Escherichia coli on the Insertion and Folding of Outer Membrane Protein A into Lipid Membranes

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PATEL, Geetika Jignesh, 2009. Kinetic Studies on the Effect of Skp and YaeT from Escherichia coli on the Insertion and Folding of Outer Membrane Protein A into Lipid Membranes

@phdthesis{Patel2009Kinet-8082, title={Kinetic Studies on the Effect of Skp and YaeT from Escherichia coli on the Insertion and Folding of Outer Membrane Protein A into Lipid Membranes}, year={2009}, author={Patel, Geetika Jignesh}, address={Konstanz}, school={Universität Konstanz} }

Biomembranes are essential for the structural and functional integrity of the cell. Membrane proteins constitute ∼ 50% of the total mass of the outer membrane in Gram-negative bacteria, and act as ion channels, drug receptors and solute transporters. Insertion and folding of membrane proteins into membranes are not well understood, but important for membrane biogenesis and cell growth. In Gram-negative bacteria, assembly of transmembrane proteins (TMPs) into the outer membrane (OM) takes place post-translationally after their translocation across the cytoplasmic membrane in unfolded form via the SecYEG translocon. Passage of outer membrane proteins (OMPs) through the periplasm is facilitated by chaperones like the seventeen kDa protein (Skp), survival factor A (SurA) and others. A multi-protein complex composed of a transmembrane protein Omp85 (YaeT in Escherichia coli) is required for assembly of OMPs into the OM. In E.coli, this complex is formed of YaeT and at least four lipoproteins, namely YfiO, YfgL, NlpB and SmpA, of which YaeT and YfiO are the only essential components. Although investigation of insertion and folding of bacterial β-barrel membrane proteins into membranes has made progress in recent years, our knowledge about the entire process is still very limited. Many questions have arisen due to the discovery of outer membrane protein (OMP) targeting and/or assembly machinery located in the periplasm as chaperones like Skp, and in the outer membrane as Omp85 complex. The factors and principles governing targeting of OMPs and the detailed mechanism by which they assemble into the OM are still unclear. Therefore, this study was performed to systematically explore the effect of these assembly factors on insertion and folding of Outer membrane protein A, which has emerged as a model protein to examine the folding mechanism of β-barrel membrane proteins into lipid membranes.<br /><br />The entire thesis work presented here is based on examining the insertion and folding of Outer membrane protein A, OmpA, from its complexes with the periplasmic chaperone, Skp into preformed lipid membranes. Role of lipid-bilayer inserted YaeT in folding of OmpA was also examined. Lastly, a method for isolation, purification and refolding of an essential lipoprotein, YfiO into lipid membranes was establishedThe major outcomes of this thesis are summarized in separate sections below.<br /><br />In the first study, spontaneous folding of OmpA of E.coli from the complex with its molecular chaperone Skp into lipid membranes (SUVs), both negatively charged (DOPC/DOPE/DOPG) and neutral (DOPC) was examined as a function of pH. This study allowed investigations on principles governing targeting and assembly of chaperone-bound OMPs into the OM.<br />In the next study, OmpA was folded into membranes (LUVs) composed of short chain phospholipids, negatively charged DLPC/DLPE/DLPG and neutral DLPC. OmpA folding was performed from urea-denatured form, its complexes with Skp in absence, and presence of LPS.<br /><br />In the third study, wild-type (wt)-YaeT, and the variant expressing its transmembrane domain (TMD-YaeT) were first purified in denatured form and then successfully refolded to their native conformations in a single step method. Next, a first in vitro assay was developed to allow further mechanistic investigations on YaeT-mediated insertion and folding of OMPs into the membrane. The effect of membrane-inserted wt-YaeT on folding of OmpA was then examined.<br />In the final study, initially a protocol for isolation of YfiO in pure form with high yield was established. YfiO was isolated with the lipid anchor upon solubilization either in chaotropic urea or a mild anionic detergent sarkosyl. Next, folding of YfiO was examined into detergent micelles and lipid bilayers by CD spectroscopy. 2009 Patel, Geetika Jignesh Kinetische Untersuchungen über den Effekt von Skp und YaeT von Escherichia Coli auf die Insertion und die Faltung von Aussenmembranprotein A in Lipid-Membranen 2012-11-30T23:25:04Z Kinetic Studies on the Effect of Skp and YaeT from Escherichia coli on the Insertion and Folding of Outer Membrane Protein A into Lipid Membranes application/pdf eng Patel, Geetika Jignesh 2011-03-24T17:39:48Z deposit-license

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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